Supporting Information

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1 Supporting Information Orally available soluble epoxide hydrolase/phosphodiesterase 4 dual inhibitor treats inflammatory pain René Blöcher 1, Karen M. Wagner 1, Raghavender R. Gopireddy 2, Todd R. Harris 1, Hao Wu 1, Bogdan Barnych 1, Sung-Hee Hwang 1, Yang K. Xiang 2, Ewgenij Proschak 3, Christophe Morisseau 1 and Bruce D. Hammock 1 1 Department of Entomology and Nematology and UC Davis Comprehensive Cancer Center, University of California Davis, One Shields Avenue, CA Davis, U.S.A. 2 Department of Pharmacology, University of California Davis, One Shields Avenue, CA 95616, Davis, U.S.A., and VA Northern California Health care system, CA Mather, U.S.A. 3 Institute of Pharmaceutical Chemistry, Goethe-University Frankfurt, Max-von-Laue-Strasse 9, D Frankfurt am Main, Germany S1

2 Table of Contents In vitro evaluation... S3 Table S1. Normalized FRET ratio change values, percent change and number of cells.... S3 Figure S1. PKA activity triggered by novel compounds using FRET biosensor in HEK293 cells. Maximal increases in PM-AKAR3 FRET ratio after stimulation with various novel compounds (1µM) or with PDE4 inhibitor Rolipram (1µM) or seh inhibitor TPPU (1µM).... S4 Figure S2. PKA activity triggered by novel compounds using FRET biosensor in HEK293 cells. Normalized Percentile maximal FRET responses of PM-AKAR3 biosensor against the increases induced by treatment with novel compounds, Rolipram and TPPU respectively.... S5 Figure S3. PKA activity triggered by novel compounds using FRET biosensor in HEK293 cells. Normalized novel compound induced dose response curves of PM-AKAR3 biosensor (IC 50, 1 at 1.9 ± 0.03 nm, 19 at 128 ± 0.05 nm, 20 at 3.9 ± 0.06 nm, 23 at 8.1 ± 0.05 nm) and compared with Rolipram (IC 50, 340 ± 0.06 nm) respectively.... S6 Pharmacokinetic evaluation... S7 Table S2. Plasma concentration (nm) for compounds given in cassette dosing.s7 Table S3. Plasma concentrations (nm) of single adminstrations of 23/MPPA.... S7 Efficacy evaluation... S8 Figure S4. Naïve rats (no induced pain state) were administered vehicle (PEG300), 3 mg/kg of MPPA or Rolipram and were assessed in a thermal withdrawal assay. The comparison in naïve rat demonstrated that Rolipram had an effect of elevating thermal withdrawal latency (TWL) in the absence of a pain state. Vehicle and MPPA administration were statistically unchanged compared to their own pretreatment scores or between treatments. The results suggest possible central nervous system activity with use of Rolipram but not MPPA. The TWL is reported as percent of baseline (pre-treatment scores normalized to 100%) with results expressed as mean ± SEM (Kruskal-Wallis One Way Analysis of Variance on Ranks, Dunn's pairwise post hoc *p 0.001, n=6-9/group).... S8 Figure S5. The open field activity assessed at 1 h post treatment distinguishes the dual inhibitor MPPA from single administration of the centrally active PDE4I Rolipram. The rats were assessed before treatment (baseline) and again at 1 h post treatment and scores are reported as percent of baseline activity. This assay demonstrated the decline in self-motivated movement with single administration of Rolipram that is not present with the vehicle PEG300 or the dual inhibitor MPPA. The small decline in score seen with the vehicle control (30%) is typical when repeating the open field from pretreatment baseline to 1 h post treatment due to a decline in the novelty of exploring the same apparatus. The results demonstrate that using this paradigm mobility decreased significantly (>50%) in Rolipram treated rats but was statistically unchanged by MPPA (One Way Analysis of Variance, Holm-Sidak pairwise post hoc, *p=0.003, n=6/group).... S9 S2

3 In vitro evaluation Table S1. Normalized FRET ratio change values, percent change and number of cells. ID mean FRET ratio change Percentage change SD Number of cells Rolipram /MPPA S3

4 TPPU Figure S1. PKA activity triggered by novel compounds using FRET biosensor in HEK293 cells. Maximal increases in PM-AKAR3 FRET ratio after stimulation with various novel compounds (1µM) or with PDE4 inhibitor Rolipram (1µM) or seh inhibitor TPPU (1µM). S4

5 Figure S2. PKA activity triggered by novel compounds using FRET biosensor in HEK293 cells. Normalized Percentile maximal FRET responses of PM-AKAR3 biosensor against the increases induced by treatment with novel compounds, Rolipram and TPPU respectively. S5

6 Figure S3. PKA activity triggered by novel compounds using FRET biosensor in HEK293 cells. Normalized novel compound induced dose response curves of PM-AKAR3 biosensor (IC 50, 1 at 1.9 ± 0.03 nm, 19 at 128 ± 0.05 nm, 20 at 3.9 ± 0.06 nm, 23 at 8.1 ± 0.05 nm) and compared with Rolipram (IC 50, 340 ± 0.06 nm) respectively. S6

7 Pharmacokinetic evaluation Table S2. Plasma concentration (nm) for compounds given in cassette dosing. Time (h) /MPPA 0 < LOD < LOD < LOD 0.25 < LOD 2 ± ± ± ± ± 13 1 < LOD 2 ± ± 12 2 < LOD 2 ± ± 6 4 < LOD 2 ± ± < LOD 1 ± 0.1 < LOD 12 < LOD 1 ± 0.2 < LOD 24 < LOD 0.3 ± 0.01 < LOD 48 < LOD < LOD < LOD Average ± SEM (n = 4) plasma concentration in rats given a cassette dose of the above compounds at 0.3 mg/kg each formulated in one solution. The cassette also contained compound 20 (0.3 mg/kg) which is not included in the table as it did not yield detectable concentrations. The limit of detection (LOD) in these measurments is 0.5 ng/ml. Table S3. Plasma concentrations (nm) of single adminstrations of 23/MPPA. Time (h) Dose 3 mg/kg 30 mg/kg 100 mg/kg 0 < LOD < LOD < LOD ± ± ± < LOD < LOD < LOD < LOD < LOD 24 < LOD < LOD < LOD 48 < LOD < LOD < LOD Average ± SEM plasma concentration at 3 mg/kg oral dose in n=4 rats. 30 and 100 mg/kg are single adminstration of 23/MPPA to one rat each and therefore do not include SEM. The limit of detection (LOD) in these measurments is 0.5 ng/ml. S7

8 Efficacy evaluation Figure S4. Naïve rats (no induced pain state) were administered vehicle (PEG300), 3 mg/kg of MPPA or Rolipram and were assessed in a thermal withdrawal assay. The comparison in naïve rat demonstrated that Rolipram had an effect of elevating thermal withdrawal latency (TWL) in the absence of a pain state. Vehicle and MPPA administration were statistically unchanged compared to their own pretreatment scores or between treatments. The results suggest possible central nervous system activity with use of Rolipram but not MPPA. The TWL is reported as percent of baseline (pre-treatment scores normalized to 100%) with results expressed as mean ± SEM (Kruskal-Wallis One Way Analysis of Variance on Ranks, Dunn's pairwise post hoc *p 0.001, n=6-9/group). S8

9 Figure S5. The open field activity assessed at 1 h post treatment distinguishes the dual inhibitor MPPA from single administration of the centrally active PDE4I Rolipram. The rats were assessed before treatment (baseline) and again at 1 h post treatment and scores are reported as percent of baseline activity. This assay demonstrated the decline in self-motivated movement with single administration of Rolipram that is not present with the vehicle PEG300 or the dual inhibitor MPPA. The small decline in score seen with the vehicle control (30% is typical when repeating the open field from pretreatment baseline to 1 h post treatment due to a decline in the novelty of exploring the same apparatus. The results demonstrate that using this paradigm mobility decreased significantly (>50%) in Rolipram treated rats but was statistically unchanged by MPPA (One Way Analysis of Variance, Holm-Sidak pairwise post hoc, *p=0.003, n=6/group). S9